This proposal addresses Thematic Area 2: Translating Basic Science Discoveries into New and Better Treatments. In particular we are proposing the development of a new human blood-brain barrier (BBB) model derived from stem cell sources amenable to high throughput assessment of central nervous system (CNS) therapeutics for their BBB permeability, a key criterion in the translation of new brain therapeutics to the clinic. A unique interdisciplinary team with expertise in blood-brain barrier, human stem cells, and brain tumor therapy was assembled to generate the preliminary feasibility data and is ready for immediate deployment on the proposed project. ABSTRACT: Millions of Americans are afflicted with neurological illnesses such as Alzheimer's disease, Parkinson's disease, and cerebral AIDS. Although significant progress has been made in the development of both small molecule pharmaceuticals and biopharmaceuticals (gene and protein medicines), very few new treatments have resulted. A major hurdle in brain drug development is the lack of robust delivery strategies that can target medicines to the brain non-invasively via the bloodstream, a process that is complicated by the presence of the blood-brain barrier (BBB) in vivo. The endothelium comprising the blood-brain barrier (BBB) is uniquely impermeable and allows only those molecules that are small (less than 500 daltons) and lipophilic to freely diffuse from the bloodstream to the interstices of the brain. Thus the endothelial barrier limits the brain uptake of the majority of small molecule pharmaceuticals (98%) and essentially prohibits the uptake of protein or gene medicines after intravenous administration. Over the past 20 years, many promising drugs have failed in clinical trials as a result of poor BBB permeability and this barrier has in part lead to the lack of new treatments and cures for brain disease. In fact, one of the most significant challenges in developing neuropharmaceuticals is the accurate prediction of BBB permeability prior to full-blown clinical trials. One potential solution is the development of cell-based in vitro models that can mimic the human BBB characteristics observed in vivo. Such models would need to be facile, scaleable and amenable to drug permeability screening for a priori prediction of brain uptake. An in vitro BBB model typically consists of a monolayer of brain endothelial cells (EC) that is grown on a permeable membrane. These cells serve as a diffusion barrier between upper and lower liquid filled compartments, the upper compartment representing the bloodstream and the lower compartment the brain. Drugs can be applied to either the upper or lower compartment to predict brain influx or efflux, respectively. Unfortunately, while in vitro BBB models can be useful in determining the transport characteristics of a candidate drug or panel of drugs, they are rarely of human origin. As a novel and timely solution, we propose creating a blood-brain barrier model based on endothelial cells derived from human pluripotent stem cells (hPSCs). As described in the preliminary data, we have devised a robust protocol for directed differentiation of human pluripotent stem cells to blood-brain barrier endothelial cells having well developed tight junctions, characteristic transporter expression, and capability to respond to cues provided by co-cultured neural cells. As such, we have approximated human BBB function in the culture dish. Thus, we propose further validation of the hPSC-derived BBB model by translating it to a variety of different pluripotent stem cell lines, and comparing the gene expression profile of stem cell-derived BBB endothelial cells to BBB endothelial cells harvested from human brain. Next, we propose to test the permeability of a panel of known drug molecules to demonstrate the model's capability of differentiating drugs that are subject to various key BBB transport mechanisms such as passive diffusion, active influx, or active efflux. Finally, the model will be used to test a panel of brain tumor therapeutics, comprised of ~100 compounds, for their human BBB permeability and post-BBB efficacy in tumor repression in vitro. Taken together, these aims have the potential to significantly alter the way CNS drugs are developed by adding a human BBB screening tool early in the development process. In this way, translation of CNS drugs from the Petri dish to the clinic may be substantially enhanced by the proposed research. PUBLIC HEALTH RELEVANCE: Developing a human BBB model with predictive capability would impact the drug development process for CNS diseases that afflict millions of people worldwide. Moreover the capability to produce large amounts of human brain endothelial cells along with NPC-derived astrocytes and neurons on demand would allow researchers and drug developers alike a new and unique resource for modeling the human BBB to pursue drug development, drug targeting, stem cell therapy, and many other endeavors that are currently intractable in humans. Finally, a detailed understanding of EGFR inhibitor permeability and efficacy using the human BBB model along with brain tumor isolates could have substantial impact on the translation of this class of medicines to the clinic.